The goal of this project is to discover multiple steps of endometrial carcinogenesis by recreating this process with human endometrial cells in vitro. Normal structure, function, and cell proliferation regulation in human endometrium involves interactions between endometrial epithelial and stromal cells and is modulated by estrogens and progestins. A "balance point" in the tissue is defined by mutual regulation by cells of each type. In endometrial carcinogenesis, this balance point is altered progressively in abnormal cells, changing the proportions and overall numbers of cells. These alterations of cell interrelations are reflected in the morphology of the abnormal tissue and are the basis of pathologic diagnostic criteria. We seek to relate these morphologic patterns to progressive alterations of cell interactions, growth regulation, and particular genes in the cells. Thus, we wish to associate a particular morphologic or functional alteration in endometrial carcinogenesis with specific gene or regulatory process alteration. In this project we will use cultures of human endometrial epithelial and stromals cells that have been partially or completely transformed by transfection of growth regulatory genes and by treatments with estrogens or chemical carcinogens to study this process. We will determine normal interactions between epithelial and stromal cells so that we will have a baseline for comparison with the altered cell functions and interactions that typify progressive steps of endometrial carcinogenesis. We will exploit our ability to cause these progressive changes with estrogens, the identified human carcinogen for endometrial cancers, in cultured human endometrial cells. We will test our hypothesis that the multiple steps of endometrial carcinogenesis represent alterations in gene function in several cell regulatory processes by examining cells at progressive stages of transformation. We will assess the intactness of the G1/S and G2/M cell cycle progression checkpoints in relation to extended cell growth capacity and immortality, and determine whether stromal cells impose checkpoint control on epithelial cells at various stages of transformation. We will assess the alteration of epithelial-stromal signalling through interleukins. We will attempt to isolate and characterize the diffusible substance produced by epithelial tumors that inhibits growth of stromal cells and causes "back-to-back" growth of tumor glands that is the diagnostic criterion for invasion. Finally we will test whether progressing cells develop autocrine growth regulation in which an epithelial growth inhibitor becomes the product of progressing tumor cells and even a growth factor for them. This may explain selective outgrowth of the most undifferentiated cancer cells. We believe that these are processes involved in progressive stages of endometrial carcinogenesis and we seek to test this hypothetical mechanism of endometrial carcinogenesis in this proposal.